The present invention relates to glycosaminoglycan derivatives useful in the inhibition of endoglycosidase activity and of tumor invasiveness or metastatic spread.
A class of biological substances called the proteoglycans form the ground substance in the extracellular matrix of connective tissues. These proteoglycans are polyanionic substances of high molecular weight and contain many different types of heteropolysaccharide side chains covalently linked to a polypeptide backbone. These proteoglycans may contain over 95% carbohydrates. The polysaccharide groups of the proteoglycans were formerly called mucopolysaccharides but now are preferably termed glycosaminoglycans since all contain derivatives of glucosamine or galactosamine.
A variety of enzymes may be involved in the normal metabolic degradation of proteoglycans. Initial proteoglycan degradation often involves proteolysis to separate or digest protein components. Such proteolysis results in the production of glycosaminoglycans. The glycosaminoglycans in turn are subject to glycosaminoglycan endoglycosidase enzymic action which produces smaller glycosaminoglycan fragments. The glycosaminoglycans or fragments thereof are subject to glycosaminoglycan exoglycosidase enzymic action which produces monosaccharides from the non-reducing ends of glycosaminoglycans.
An increasing interest in the endoglycosidases has arisen in recent years because of a possible relationship of these enzymes with tumor invasiveness and tumor metastatic activity. Nicolson (1982, Biochem. Biophys. Acta. V 695, pp 113-176) reviewed a variety of oligosaccharide-degrading enzymes (pp 141-142) reported to be of interest in malignant disease. Nicolson (1982, J. Histochem. & Cytochem. V 30, pp 214-220) described a proposed mechanism for tumor cell invasion of endothelial cell basal lamina and a related production of degradation products from proteins and glycosaminoglycans. Kramer et al., (1982, J. Biol. Chem. V 257, pp 2678-2686) reported a tumor-derived glycosidase capable of cleaving specifically glycosaminoglycans and releasing heparan sulfate-rich fragments.
Irimura et al., (1983a, Analyt. Biochem. V 130, pp 461-468) describe high-speed gel-permeation chromatography of glycosaminoglycans. Heparan sulfate degrading activity of melanoma cells was measured by using this chromatographic procedure. Nakajima et al., (1983, Science, V 220, pp 611-613) described a relationship of metastatic activity and heparan sulfate degrading activity in melanoma cell lines. The disappearance of higher molecular weight heparan sulfate was followed by polyacrylamide gel electrophoresis, staining and densitometry.
Vlodavsky et al., (1983, Cancer Res. V 43, pp 2704-2711) described the degradation by two T-lymphoma cell lines of .sup.35 S labeled proteoglycans from confluent endothelial cells. The highly metastatic line had much higher .sup.35 S liberating activity than did the low metastatic line.
Irimura et al., (1983c, Proc. Am. Soc. Cancer Res. V 24, p 37, abstract 144), using high performance liquid chromatography, describe heparan sulfate degradative enzyme activity of melanoma cells. Nakajima et al., (1984, J. Biol. Chem. V 259, pp 2283-2290) describe characterizations of metastatic melanoma heparanase. High speed gel permeation chromatography and chemical analyses were used in a description of functional substrates and products formed. Nakajima et al. (1986, Anal. Biochem., in press) synthesized a solid-phase substrate for heparanase by crosslinking radiolabeled and reductively aminated HS to amino-reactive agarose beads via one covalent linkage. This solid-phase substrate was used for the measurement of heparanase activity in various human melanoma cell lines (Nakajima et al., (1986) Cancer Letters, V 31, pp 277-283) and sera from mammary adenocarcinoma-bearing rats and malignant melanoma patients (Nakajima et al., (1986) In: Cancer Metastasis: Experimental and Clinical Strategies, D. R. Welch, B. K. Bhuyan, L. A. Liotta, eds. Alan R. Liss, Inc., New York, pp 113-122).
From the foregoing it may be seen that significant interest exists in convenient, accurate and reproducible endoglycosidase assays and production of potent heparanase inhibitors, particularly since endoglycosidases may play critical roles in the establishment of tumor metastases.
The ability of tumor cells to invade host tissues and metastasize to distant, often specific organ sites, is one of their most important properties. Metastasis formation occurs via a complex series of unique interactions between tumor cells and normal host tissues and cells. These processes involve several discrete and selective steps such as: invasion of surrounding tissues, penetration of lymphatics of blood vessels and transport in lymph or blood, or dissemination into a serous cavity, arrest and invasion at distant sites, and survival and growth to form secondary lesions.
Basement membranes are continuous sheets of extra-cellular matrix composed of collagenous and non-collagenous proteins and proteoglycans that separate parenchymal cells from underlying interstitial connective tissue. They have characteristic permeabilities and play a role in maintaining tissue architecture. Metastasizing tumor cells must penetrate epithelial and endothelial basement membranes during invasion and metastasis, and the penetration and destruction of basement membranes by invasive tumor cells has been observed using electron microscopy. Since basement membranes are rigid structures formed from unique sets of macromolecules, including type IV collagen, laminin, heparan sulfate (HS), proteoglycan and fibronectin, the successful penetration of a basement membrane barrier probably requires the active participation of more than one tumor cell-associated enzyme.
Due to its unique physical and chemical properties such as its polyanionic character and barrier properties against macromolecules (Kanwar et al., 1980 J. Cell. Biol. V 86, pp 688-693), HS is an important structural component of basement membranes. HS binds to fibronectin, laminin and type IV collagen, and these molecules have been collectively observed in the basal lamina using antibodies raised against each component. HS may be involved in basal lamina matrix assembly by promoting the interactions of collagenous and non-collagenous protein components while protecting them against proteolytic attack. Thus, the destruction of HS proteoglycan barrier could be important in basement membrane invasion by tumor cells.
The interactions between malignant cells and vascular endothelium have been studied using monolayers of cultured vascular endothelial cells that synthesize an extracellular matrix resembling a basement membrane. With this model, it has been found that metastatic B16 melanoma cells degrade matrix glycoproteins, such as fibronectin, and matrix sulfated glycosaminoglycans, such as heparan sulfate. Since HS was released in solution as fragments approximately one-third their original size, it has been proposed that metastatic tumor cells characteristically have a HS endoglycosidase.
The relation between metastatic properties and the ability of five B16 melanoma sublines of various implantation and invasion characteristics to enzymatically degrade subendothelial extracellular matrix indicated that highly invasive and metastatic B16 sublines degraded sulfated glycosaminoglycans faster than did sublines of lower metastatic potential (Nakajima et al., (1983), Science V 220, p 611), and intact B16 cells (or their cell-free homogenates) with a high potential for lung colonization also degraded purified heparan sulfate at higher rates than did B16 cells with a poor potential for lung colonization (ibid).
The abilities of B16 cells to degrade HS from various origins and other purified glycosaminoglycans (heparin, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid) have been studied. In order to analyze glycosaminoglycan degradation products, an analytic procedure was developed using high-speed gel permeation chromatography (Irimura et al., (1983a) Anal. Biochem. V 130, p 161; Nakajima et al., (1984) J. Biol. Chem. V 259, p 2283). HS metabolically labeled with .sup.35 S-sulfate was purified from basement membrane producing EHS sarcoma and PYS-2 carcinoma cells, and subendothelial matrices of bovine aortic endothelial (BAE) and corneal endothelial (BCE) cells (ibid). HS molecules purified from bovine lung and other glycosaminoglycans were labeled with tritium at their reducing termini using .sup.3 H-NaBH.sub.4. These labeled glycosaminoglycans were incubated with B16 cell extracts in the absence or presence of D-saccharic acid 1,4-lactone, a potent exobeta-glucuronidase inhibitor, and degradation fragments were analyzed by high-speed gel permeation chromatography.
HS isolated from the various origins described above were all degraded into fragments of characteristic molecular weight, in contrast to hyaluronic acid, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan sulfate, keratan sulfate, and heparin, which were essentially undegraded. Heparin, but not other glycosaminoglycans, inhibited HS degradation. The time dependence of HS degradation into particular molecular weight fragments indicated that melanoma heparanase cleaves HS at specific intrachain sites (ibid). In order to determine specific HS cleavage points, the newly formed reducing termini of HS fragments were investigated by: labeling with .sup.3 H-NaBH.sub.4 ; hydrolysis to monosaccharides; and analysis of these saccharides by paper chromatography. Since .sup.3 H-reduced terminal monosaccharides from HS fragments were overwhelmingly (&gt;90%) L-gulonic acid, the HS-degrading enzyme responsible was an endoglucuronidase (heparanase).
HS-degrading endoglucuronidases have been found in various tissues, such as human skin fibroblasts, rat liver cells, human placenta, and human platelets. HS-degrading endoglucuronidases in mammalian cells were reported previously by other investigators to be "heparitinases" to indicate heparitin sulfate (heparan sulfate)-specific endoglycosidase. However, heparitinase originally was used to designate an elimination enzyme (EC 4.2.2.8) in Flavobacterium heparinum, and this enzyme cleaves non-sulfate and monosulfated 2-acetoamido-2-deoxy-alpha-D-glucosyl-D-hexuronic acid linkages of HS. Since HS-specific endoglycosidases in mammalian cells are endoglucuronidases, except for one found in skin fibroblasts, it was proposed that mammalian cell endoglucuronidases capable of degrading HS should be called "heparanases", consistent with the currently used term "heparan sulfate".
High heparanase activity in human melanoma cells was demonstrated using a solid-phase substrate, partially N-desulfated N-[.sup.14 C] acetylated HS crosslinked to agarose beads via one covalent linkage (Nakajima et al., (1986) Cancer Letters, V 31, pp 277-283; Nakajima et al., (1986) Anal. Biochem. in press). All of the 15 human melanoma cell lines tested were found to have heparanase activity and almost all possessed high activities comparable or greater than that of the murine B16-F1 melanoma line. Human A375 melanoma variants of high lung metastatic potential in athymic nude mice had significantly higher heparanase activities than did A375 parental cells of low metastatic potential.
High heparanase activity was also found in the sera from highly metastatic tumor-bearing animals and malignant melanoma patients (Nakajima et al, (1986) In: Cancer Metastasis: Experimental and Clinical Stategies, D. R. Welch, B. K. Bhuyan, L. A. Liotta, eds., Alan R. Liss, Inc., New York, pp 113-122). A significant difference in serum heparanase activity was found between a group of normal adults and a group of malignant melanoma patients (N=35, p&lt;0.05). The mean values of serum heparanase activities in normal adults and malignant melanoma patients were 3.97 and 9.43 mg HS/h/ml serum, respectively. Some of the patients having documented lymph node metastases had 4 to 6-fold higher serum heparanase activities than normal adults.
Anticoagulants, such as heparin, warfarin, dextran sulfate and Ancrod; (Agkistrodon rhodostoma venom protease, Abbott) have been used to prevent blood coagulation and reduce the formation of metastatic tumor cell thrombi in the blood which lodge more effectively in the microcirculation Hilgard et al., Eur. J. Cancer, V 12, pp 755-762, 1976). H. Ludwig (Gynakologe, V 7, pp 1-10, 1974) reported longer recurrence-free intervals and less metastases in patients during irradiation of their gynaecological cancers. Heparin has been used as an adjuvant therapy agent with combination chemotherapy of inoperable lung cancer with enhanced therapeutic effects (Elias, Proc. Amer. Assoc. Cancer Res., V 14, p 26, 1973, Abst.). L. Michaels (Lancet, V 2, pp 832-83, 1964) found that the cancer incidence and death rate was lower than expected for patients receiving heparin. Dextran sulfate (M.sub.r --7,000) has been used to inhibit metastasis of rat lung tumors, and human lung cancer patients have received long term oral dextran sulfate. Although the antimetastatic effects of dextran sulfate were marginal in cancer patients, this was probably due to the low intestinal absorption of dextran sulfate (Suemasu, Gann Monogr., V 20. pp 163-172, 1977).
Heparin and related sulfated glycoconjugates with anticoagulation properties, such as dextran sulfate have been used experimentally as antimetastatic agents (Tsubura et al., (1977) Gann Monogr. Cancer Res., V 20, pp 147-153; Hilgard, (1984) in Cancer Invasion and Metastasis, Biologic and Therapeutic Aspects, Nicolson et al., eds, pp 353-360 Raven Press, N.Y.) The basis for this use was the assumption that platelet aggregation, together with activation of the coagulation cascade, enhanced the formation of tumor embolism and increased implantation and metastatic colonization of blood-borne tumor cells. In other studies on the effects of heparin on metastasis, however, heparin administration increased, decreased, or had no effect on tumor cell dissemination and organ colonization, depending on the experimental system. Mechanisms other than the anticoagulation effects of heparin on tumor metastasis were suggested by these results, but the possible involvement of tumor heparanase had not been considered. The present invention relates to heparin derivatives without anticoagulant properties and which inhibit the heparanase activity of metastatic mouse melanoma cells. These substances were useful as tools for in vitro and in vivo studies involving the role of heparanase in tumor invasion and metastasis.
Using oral administration of heparin in combination with hydrocortisone, it was reported that complete regression of established transplantable tumors in mice could occur through inhibition of tumor angiogenesis (Folkman et al., Science, V 221, pp 719-725, 1983). This suggested that anti-angiogenic substances could be used for cancer therapy (J. Folkman in: Important Advances in Oncology, de Vita et al., eds, pp 42-62, J. B. Lippincott, Philadelphia, 1985). In such studies heparin was administered in the drinking water of animals. For example, hamsters have been inoculated with transplantable pancreatic carcinoma cells and have been treated by receiving heparin or hexuronyl hexaminoglycan sulfate, a heparin derivative, in their drinking water at concentrations of 10 mg/ml with hydrocortisone (0.5 mg/ml). After treatment for 6-9 days, the tumors were examined. In 3 out of 4 of the tumors the hexuronyl hexaminoglycan sulfate plus hydrocortisone resulted in significant reductions in growth rate in vivo and significant inhibitions of capillary endothelial cell migration in Boyden chambers in vitro (Rong et al., Cancer, V 57, pp 586-590, 1986).
Proliferation of vascular smooth muscle cells has been shown to be an important step in the pathogenesis of arteriosclerosis (Ross et al., Science, V 180, pp 1332-1339, 1973). Commercial heparin preparations have been separated into anticoagulant and non-anticoagulant mixtures by use of antithrombin to remove the anticoagulant heparin. Both of these forms of heparin significantly inhibit the growth of smooth muscle cells in vitro (Hoover et al., Circulation Res., V 47, pp 578-583, 1983). Using the heparin preparations at a concentration of 10 ug/ml resulted in approximately 50% inhibition of .sup.3 H-thymidine uptake by arterial smooth muscle cells, which is indicative of growth inhibition. Administration of anticoagulating and non-anticoagulating heparin fractions inhibited intimal smooth muscle proliferation, as determined by the total plaque volume two weeks after arterial injury. Non-anticoagulating heparin given at a dose of 100 USP units per kg body weight per hour in Sprague-Dawley rats resulted in 77% inhibition of myointimal growth (Guyton et al., Circulation Res., V 46, pp 625-634, 1980).
Further studies indicated that the minimum fragment size of heparin, which was growth inhibitory toward vascular smooth muscle cells, was a hexasaccharide, and the maximum anti-proliferative activity was obtained with a 12-residue heparin. Most modified heparins (totally desulfated, N-desulfated, and totally desulfated re-N-acetylated) lost their anti-proliferative activity, but the N-desulfated N-resulfated heparin and the N-desulfated N-acetylated heparin retained full growth inhibitory properties (Castellot et al., J. Cell. Physiol., V 120. pp 315-320, 1984). Diseases, such as arteriosclerosis, develop over a long period of time. Therefore, the main use of such treatments might be in vascular damage due to trauma or surgery, such as artery vein grafts or arteriovenous shunts for kidney dialysis.